ARTICLE

Smart Human Serum Albumin-

Indocyanine Green Nanoparticles
Generated by Programmed Assembly
for Dual-Modal Imaging-Guided
Cancer Synergistic Phototherapy
Zonghai Sheng,†,§ Dehong Hu,†,§ Mingbin Zheng,† Pengfei Zhao,† Huilong Liu,‡ Duyang Gao,† Ping Gong,†
Guanhui Gao,† Pengfei Zhang,† Yifan Ma,*,† and Lintao Cai*,†
†
Guangdong Key Laboratory of Nanomedicine, CAS Key Laboratory of Health Informatics, Shenzhen Institutes of Advanced Technology, Chinese Academy of
Sciences, Shenzhen 518055, PR China and ‡Department of Oncology, General Hospital of Beijing Command, PLA, Beijing 100700, PR China. §Z. S. and D. H.
contributed equally to this paper.

ABSTRACT Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT),
is a light-activated local treatment modality that is under intensive preclinical and clinical
investigations for cancer. To enhance the treatment efficiency of phototherapy and reduce the
light-associated side effects, it is highly desirable to improve drug accumulation and precision guided
phototherapy for efficient conversion of the absorbed light energy to reactive oxygen species (ROS) and
local hyperthermia. In the present study, a programmed assembly strategy was developed for the
preparation of human serum albumin (HSA)-indocyanine green (ICG) nanoparticles (HSA-ICG NPs) by
intermolecular disulfide conjugations. This study indicated that HSA-ICG NPs had a high accumulation with tumor-to-normal tissue ratio of 36.12 ( 5.12 at
24 h and a long-term retention with more than 7 days in 4T1 tumor-bearing mice, where the tumor and its margin, normal tissue were clearly identified via
ICG-based in vivo near-infrared (NIR) fluorescence and photoacoustic dual-modal imaging and spectrum-resolved technology. Meanwhile, HSA-ICG NPs
efficiently induced ROS and local hyperthermia simultaneously for synergetic PDT/PTT treatments under a single NIR laser irradiation. After an intravenous
injection of HSA-ICG NPs followed by imaging-guided precision phototherapy (808 nm, 0.8 W/cm2 for 5 min), the tumor was completely suppressed, no
tumor recurrence and treatments-induced toxicity were observed. The results suggest that HSA-ICG NPs generated by programmed assembly as smart
theranostic nanoplatforms are highly potential for imaging-guided cancer phototherapy with PDT/PTT synergistic effects.

KEYWORDS: indocyanine green . theranostics . photothermal therapy . photodynamic therapy . synergistic effect .
photoacoustic imaging

P
hototherapy, represented by photo-
dynamic therapy (PDT) and photo-
thermal therapy (PTT), is a non-
invasive and effective approach for cancer
treatment.1,2 This type of light-triggered
treatment modality has remarkably im-
proved selectivity and fewer side effects as
compared to conventional radiotherapies
and chemotherapies.3 In phototherapy, light
is administered, absorbed by the photosen-
sitizer (PS) or photothermal agent, and con-
verted into reactive oxygen species (ROS) or
local hyperthermia, leading to tumor cell
death.4 To selectively and efficiently destroy
the targeted cancer cells while sparing nor-
mal tissue, sufficiently enhancing the tumor
accumulation of phototherapeutic agent,
precisely focusing the laser beam on the
tumor areas, and adequately converting the
absorbed light energy to ROS or heat are
important.5
7 At present, several types of
imaging technologies, including computed
tomography (CT),8 positron emission tomog-
raphy (PET),9 magnetic resonance imaging * Address correspondence to
(MRI),10
12 ultrasonic imaging13 and photo- lt.cai@siat.ac.cn,
yf.ma@siat.ac.cn.
acoustic imaging (PA)14 have been applied
as an assistance tool for guiding cancer Received for review August 18, 2014
phototherapy. However, real-time and pre- and accepted December 2, 2014.
cise control of the laser irradiation scope
Published online December 02, 2014
remain a major challenge as treatment 10.1021/nn5062386
required.15 Thus, during laser treatment, it
is necessary to real-time outline the tumor C 2014 American Chemical Society

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 ARTICLE
Figure 1. Schematic illustration of HSA-ICG NPs for in vivo dual-modal imaging, tumor margin detection, and simultaneous
PDT/PTT treatments. Uptake of HSA-ICG NPs is presumably mediated by EPR effect (passive targeting) and the gp60
transcytosis pathway (active targeting) and subsequent binding to SPARC (secreted protein acidic and rich in cysteine, SPARC)
in the tumor cells. The tumor, tumor margin and normal tissue could be detected using in vivo NIR and PA dual-modal imaging
and spectrum-resolved technology. Upon the single NIR laser irradiation, the HSA-ICG NPs can simultaneously convert the
absorbed light energy to ROS and heat for synergistic PDT/PTT treatments.

and its margin, normal tissue, which is prone to pre-
cisely focus the laser beam on the tumor areas. Fluo-
rescence (FL) imaging in near-infrared (NIR) window
(650
900 nm) holds much promise due to minimal
autofluorescence and tissue scattering.16 It not only
could real-time monitor in vivo dynamic distribution of
PS or photothermal agent, but also can detect the
tumor and its margin, normal tissue using contrast
enhancement and spectrum-resolved technology.17 In
addition, photoacoustic (PA) imaging has a high spatial
resolution of optical imaging with the high penetration
depth of ultrasound imaging, which can overcome
some limits of NIR FL imaging.18 Therefore, in vivo
NIR FL and PA dual-modal imaging combined with
spectrum-resolved technology would provide a real-
time, sensitive, noninvasive assistant tool for cancer
phototherapy.
In addition, to enhance the treatment efficiency and
fully exert the synergistic effect of PDT and PTT, the PS
and photothermal agent need to be simultaneously
delivered to tumor in a site-specific manner.19
22
Recent studies mainly focused on the PS-photothermal
agent composite nanostructures for simultaneous
PDT/PTT therapy triggered by a single NIR laser.23,24
For instance, Wang et al. prepared photosensitizer-
conjugated gold nanostars which could be induced
both PDT and PTT effects by single wavelength NIR
laser, and obtaining improved cancer therapy efficacy
in MDA-MB-435 tumor bearing mice.23 Lin et al. con-
firmed that photosensitizer-loaded plasmonic vesicu-
lar assemblies of gold nanoparticles (NPs) simulta-
neously excited by 671 nm laser for PDT/PTT treat-
ments, which subsequently could significantly reduce
tumor growth in vivo.24 The unique treatment im-
proved the therapeutic efficacy, reduced operation
steps and time. However, the simultaneous synergistic
therapy strongly depended on the overlapped both
optical absorption of PS and photothermal agent. The
particular requirement and complicated synthesis pro-
cess could limit its further applications.
Indocyanine green (ICG) is a NIR dye for clinic
approved by the U.S. Food and Drug Administration
(FDA). It not only can be used for NIR FL and PA
imaging, but also can convert the absorbed light
energy to ROS and local hyperthermia for PDT and
PTT, respectively.25 Therefore, ICG could be considered
as a kind of ideal theranostic platform for biomedi-
cal applications. Recently, several research groups
including ours have developed some exogenous nano-
carriers, such as polyallylamine,26 calcium phos-
phosilicate,27
29 lipid
polymer,30
33 and perfluoro-
carbon,34
37 to prepare ICG NPs for improving stability
and tumor-specificity. However, the tumor could
be partially inhibited by the single photothermal or

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Figure 2. The synthesis and characterization of HSA-ICG NPs. (a) Schematic illustration of the programmed assembly strategy
for the preparation of HSA-ICG NPs. (b) TEM image of HSA-ICG NPs, which were negatively stained with 2% phosphotungstic
acid. (c) Size distribution of HSA-ICG NPs. (d) Time-dependent temperature and (e) singlet oxygen generation of HSA-ICG NPs
during single NIR laser irradiation (808 nm laser, light dose rate: 1.0 W/cm2), CICG = 0.2 mg/mL.

photodynamic effect of ICG.29,31 Herein, a programmed
assembly strategy was developed for fabricating ICG
NPs using endogenous human serum albumin (HSA) as
delivery system, applied for in vivo NIR FL and PA dual-
modal imaging, tumor margin detection and simulta-
neous synergistic PDT/PTT therapy (Figure 1). The tumor
could be completely ablated, and on tumor recurrence
was observed. The smart HSA-ICG NPs have some
unique advantages. (1) Biosafety: as an endogenous
protein approved by FDA for intravenous administration,
HSA as ICG delivery system is nontoxic, nonantigenic,
and biodegradable.38,39 (2) Tumour targeting: HSA NPs
possess both passive and active tumor-targeting abilities
via enhanced permeability and retention (EPR) effect
and gp60 and SPARC receptor-mediated transcytosis,
respectively.40 (3) Stimuli response: HSA NPs are pre-
pared with intermolecular cross-linking by disulfide
bond, which exhibit excellent reductive-sensitive
activity.41 (4) Theranostics: HSA-ICG NPs can be used
for tumor imaging and its margin detection, and syner-
gistic PDT/PTT treatments.25
RESULTS AND DISCUSSION
Synthesis and Characterization of the HSA-ICG NPs. HSA is
the most abundant plasma protein. Each HSA molec-
ular is composed of one sulfhydryl group and 17 pair of
disulfide bonds.39 In this study, the HSA was incubated
with excessive glutathione (GSH), an endogenous re-
ducing agent, and these disulfide bonds of HSA mo-
lecular were cleaved. The obtained free sulfhydryl
groups were assembled again by intermolecular disul-
fide bonds and formed HSA NPs (Figure 2a). No
exogenous cross-linkers and toxic chemicals were
involved during the preparation process. To verify
our hypothesis, the changes of the total number of
sulfhydryl group of HSA were first monitored with
Ellman's method.42 As HSA was incubated with exces-
sive GSH, the average number of free sulfhydryl in each

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Figure 3. Cell uptake of free ICG and HSA-ICG NPs. (a) Confocal FL images of 4T1 and 293T subcellular localization of free ICG
and HSA-ICG NPs after 3 h incubation. Blue represented FL of DAPI, and red represented FL of ICG. (b) Flow cytometric analysis
of mean FL intensity (n = 10 000 cells) in 4T1 cells incubated with media (black), free ICG (red), HSA-ICG NPs with HSA blocking
(blue) and HSA-ICG NPs (purple) for 3 h.
HSA molecular increased from 0.45 to 4.6, then decreased
to 0.5 after formation of HSA-ICG NPs (Figure S1,
Supporting Information). These varies of the number
of sulfhydryl group indicated the formation process of
intermolecular disulfide bonds, which are prone to
stabilize the HSA-ICG NPs and make the NPs have
reductive-sensitive activity. The circular dichroism
(CD) spectra analysis was conducted to examine the
characteristic of HSA's secondary structures (Figure S2).
The results illustrated that the absorbance of HSA NPs
in the range from 190 to 255 nm was almost same as
natural HSA, indicating that the secondary structuring
of HSA, including R helix, β sheet and random coil, did
not change during the whole process.43
The size of HSA-ICG NPs could be turned by chang-
ing the amount of ethanol (Figure S3). Considering the
size distribution and ICG loading efficiency, the opti-
mized assembly approach was used in following ex-
periments. As shown in Figure 2b, the obtained HSA-
ICG NPs dispersed as individual NPs with a well-defined
spherical shape and homogeneously distributed with
diameter ranging from 25 to 35 nm. The average
hydrodynamic diameter of HSA-ICG NPs was 75 (
2.4 nm via DLS measurement (Figure 2c), which was
larger than corresponding TEM results. The reason was
that DLS showed an average hydrodynamic particle
size, while TEM images detected the dehydration
morphology of HSA-ICG NPs. When GSH (50 mM) was
added into HSA-ICG NPs, the average hydrodynamic
size of HSA-ICG NPs was decreased to 8.0 ( 0.6 nm,
which indicated that prepared HSA-ICG NPs had ex-
cellent reductive-sensitive activity (Figure 2c). The zeta
potential of HSA-ICG NPs was
39.0 ( 0.7 mV (Figure S4).
The content of ICG in HSA NPs was 11.0% determined
by UV/vis absorption spectra. The encapsulation of
ICG in HSA NPs prevented its interaction with the
SHENG ET AL.
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surrounding environment and delayed its decomposi-
tion thus improving the photothermal and colloid
stability and biocompatibility of ICG (Figure S5
S7).
Upon the NIR laser irradiation, the temperature of HSA-
ICG NPs increased to 59.4 °C, while the PBS only
increased to 26.9 °C (Figure 2d). The results suggested
that HSA-ICG NPs could cause significant hyperther-
mia, leading to an irreversible damage to tumor cells
(above 42 °C).2 The process for photothermal response
of HSA-ICG NPs was accompanied simultaneously by
generation of ROS. As showing Figure 2e, the FL inten-
sity of singlet oxygen sensor (20 ,70 -dichlorofluorescin
diacetate, Figure S8) generation from HSA-ICG NPs
increased with prolonging the laser irradiation time,
indicating continuous generation of singlet oxygen
by HSA-ICG NPs. The above results indicated that
HSA-ICG NPs could be used as a photothermal and
photodynamic agent for simultaneous PDT/PTT ther-
apy triggered by single NIR laser. Moreover, the gen-
eration of singlet oxygen of HSA-ICG NPs enhanced
with temperature rise (Figure S9). The result exhibited
the photothermal effect mediated by HSA-ICG NPs was
benefit for PDT enhancement.44 Therefore, the simul-
taneous synergistic PDT/PTT effect induced by HSA-
ICG NPs with single NIR laser could adequately convert
the NIR laser energy to ROS and local hyperthermia for
enhanced phototherapy.
In Vitro Cell Uptake and Synergistic Phototherapy. The cell
uptake behavior of free ICG and HSA-ICG NPs was in-
vestigated through confocal microscopy (Figure 3a).
After 3 h incubation, a little amount of free ICG was
observed in 4T1 breast cancer cells. In contrast, a large
number of HSA-ICG NPs entered into cancer cells and
localized in the cytoplasm. On the contrary, when the
HSA-ICG NPs incubated with normal cells (293T human
kidney cells) under the same conditions, low FL signal
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Figure 4. In vitro phototherapy. (a) FL image of 4T1 cells after PDT, PTT and simultaneous PDT/PTT treatments. Viable cells
were stained green with calcein-AM, and dead/later apoptosis cells were floating and eluted, or stained red with PI, CICG =
2.5 μg/mL. (b) Quantitative detection of 4T1 cells viability following PDT, PTT and simultaneous PDT/PTT treatments for 3 h.
The date are shown as mean ( SD (n = 3). (c) Flow cytometry analysis of 4T1 cells after PDT, PTT and simultaneous PDT/PTT
treatments. Positive PI and Annexin V-Alexa Fluor 488 cells were defined as late apoptosis/necrotic cells. CICG = 2.5 μg/mL.

was observed in the cytoplasm (Figure 3a). To further
demonstrate the active tumor-targeting ability of HSA-
ICG NPs, the competition binding experiments using
HSA were performed. A large number of HSA-ICG NPs
entered into 4T1 cancer cells compared to the HSA-ICG
NPs with blocking group (Figure 3a). The quantitative
results of flow cytometric analysis showed that the
average FL intensity was 10.3-fold greater in HSA-ICG
NPs group than that in HSA-ICG NPs with HSA blocking
group (Figure 3b). The results demonstrated that the
encapsulation of ICG in HSA NPs could improve cell
uptake ability, and enhance cancer cell targeting. The
reasons probably were due to the presence of albumin
receptor, SPARC (Secreted Protein, Acidic and Rich in
Cysteine), in 4T1 cells. HSA-binding with SPARC and
subsequent uptake by tumor cells increased its accu-
mulation in cancer cells.39 The enhanced cell uptake of
HSA-ICG NPs was beneficial for improving the photo-
therapeutic efficiency.
Under the NIR laser irradiation, the HSA-ICG NPs
in cancer cells could induce ROS and local hyperther-
mia generation, which can be detected using DHE
probe and an infrared thermal camera, respectively
(Figure S10
S11). In order to visually evaluate the
in vitro therapeutic effect of HSA-ICG NPs, the cells were
stained with calcein-AM and propidium iodide (PI) to
identify live and dead/late apoptotic cells, respectively.
As shown in Figure 4a, cells all displayed green FL in
the control group, which suggested that pure laser
irradiation alone cannot kill cells. To achieve single PDT
and PTT treatments, we maintained a constant tem-
perature (25 °C) or treated cells with 100 mM NaN3, a
well-known 1O2 scavenger, in order to avoid producing
singlet oxygen during laser irradiation.21 These results
illustrated that some cells were killed and exhibited red
FL in the single PDT and PTT treated groups. On the
contrary, without any post-treatments, HSA-ICG NPs
provided simultaneously synergetic PDT/PTT to induce
most of cells death and exhibiting intense homo-
geneous red FL. The quantitative treatment effect of
HSA-ICG NPs was examined by MTT assay on 4T1 cells.
As shown in Figure 4b, single PDT and PTT caused 57.5
and 84.4% of cell dead, respectively. The simultaneous
PDT/PTT treatments further induced cell death up to
92.8%. The results were further validated by flow
cytometry (Figure 4c). The 4T1 cells were treated with
PDT, PTT, and simultaneous PDT/PTT, then labeled with
PI and Annexin V-Alexa Fluor 488, respectively. The
results showed that simultaneous PDT/PTT treatment
more significantly induced 4T1 cell late apoptosis/
necrosis (94.4%) as compared with single PDT
(49.3%) or PTT (71.9%). It suggested that the simulta-
neous synergistic PDT/PTT effect was responsible for
the improved therapeutic efficiency of HSA-ICG NPs.
In Vivo Dual-Modal Imaging and Tumour Margin Detection.
The feasibility of HSA-ICG NPs for in vivo NIR FL and PA
dual-modal imaging and tumor margin detection was
investigated. Athymic nude mice with subcutaneous

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Figure 5. In vivo FL imaging of nude mice bearing 4T1 tumors after i.v. injection of free ICG and HSA-ICG NPs, respectively.
In vivo FL imaging results of (a) free ICG and (b) HSA-ICG NPs at different time intervals. (c) Ex vivo FL images of major organs
and tumors after injection of free ICG and HSA-ICG NPs at 24 h. (d) Biodistribution of free ICG and HSA-ICG NPs in mice
determined by the ICG FL from diluted tissue lysates. The data are shown as mean ( SD (n = 3).

4T1 breast cancer xenografts were used as the animal
modal. When the size of tumors reached ∼60 mm3, the
mice were intravenously injected with free ICG and
HSA-ICG NPs, respectively, and acquired FL images on
CRI maestro system using a 704 nm excitation wave-
length and a 735 nm filter at different time points. As
shown in Figure 5a, in the case of free ICG-treated mice,
the FL signals distributed extensively in liver tissue and
less in tumor tissue after 0.5 h postinjection, and
completely disappeared after 24 h postinjection. On
the contrary, the FL signal of tumor tissue strength-
ened with the increase of time interval, and reached a
peak after 24 h postinjection of HSA-ICG NPs
(Figure 5b). After 7 days, the FL signal in tumor tissue
still could be detected. The pharmacokinetic data
showed that the blood circulation half-life of HSA-ICG
NPs was significantly prolonged compared to free ICG
(t1/2 HSA-ICG NPs =2.86 h vs t1/2 free ICG =0.12 h),
indicating the long blood circulation time of HSA-ICG
NPs (Figure S12). The ex vivo images of major organs
and tumors demonstrated that most of ICG accumu-
lated in the liver 24 h after i.v. injection of free ICG. The
HSA NPs dramatically increased the accumulation of
ICG in tumor, followed by liver and kidneys (Figure 5c).
The HSA-ICG NPs biodistribution in various tissues was
detected, and expressed as percentage of dose per
unit mass of tissue (% ID/g). As shown in Figure 5d, at
24 h postinjection, the ICG content in tumor reaches
maximum of about 28.8% dose/g tissue, and tumor-to-
normal tissue ratio is 36.12 ( 5.12, suggesting high
tumor accumulation ratio.
As a dual-modal contrast agent, the HSA-ICG NPs
exhibited an excellent property of PA imaging (Figure
S13). As showing Figure 6a, before i.v. injection of free
ICG and HSA-ICG NPs, only large blood vessels in tumor
could be visualized using hemoglobin as an endogen-
ous contrast agent.4 After i.v. injection of free ICG and
HSA-ICG NPs, the PA signals at tumor tissue increased
over time (Figure 6b). The HSA-ICG NPs exhibited
stronger PA signal than that of free ICG, which agreed
with the in vivo NIR FL imaging behavior. The enhanced
PA signals after 24 h postinjection visually revealed the
uniform distribution of ICG within and outside the
tumor microvessels. The results showed that HSA-ICG
NPs-based PA imaging provided a high spatial resolu-
tion, which is prone to understand the distribution of
ICG in tumor tissue and get the information on tumor
microstructure.
The real-time, sensitive and long-term NIR FL and
PA dual-modal imaging is useful in confirming tumor

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Figure 6. In vivo tumor PA imaging. (a) PA imaging of the mice bearing 4T1 tumor injected with free ICG and HSA-ICG NPs at
different times. (b) The mean PA intensity of tumor tissue at 0, 1, 6, 12, and 24 h postinjection, respectively. The data are shown
as mean ( SD (n = 3).

Figure 7. In vivo tumor margin detection. (a) Photograph showing a laser beam focusing on the tumor site. In vivo FL spectra
were obtained from the tumor site, tumor margin, and normal tissue at 785 nm excitation. (b) Photograph of tumor, tumor
margin and normal tissue labeled with different numbers. (c) Averaged FL spectra of the tumor site, tumor margin and normal
tissue. (d) Tumour mapping imaging with integrated FL intensity of HSA-ICG NPs at various locations.

location, detecting tumor large blood vessels and
optimizing the content of phototherapeutic agents.14
However, scattering of light in tumor tissue severely
impedes imaging of tumor margin.15 Therefore, it is
essential to identify the tumor margin for planning
spatially localized phototherapy. For proof-of-concept,
ICG-based FL spectrum-resolved technology provided
a promising tool for highly sensitive detection of tu-
mor boundary.17 785 nm NIR laser was carried out as
an excitation wavelength, and the in vivo emission

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Figure 8. In vivo cancer phototherapy in xenograft mice models with 4T1 breast cancer. Schematic illustration shows the
tumor tissue was irradiated by NIR laser beam with three different ways: (a) part irradiation, (b) accurate irradiation, (c) over
irradiation. (d) IR thermal images of 4T1 tumor-bearing mice exposed to 808 nm laser for 5 min (0.8 W/cm2). (e) H&E stained
images of tumor sections collected from different treated groups of mice 4 h post treatment. (f) Tumour growth curves of
different groups of 4T1 tumor-bearing mice. (g) Survival rates of mice bearing 4T1 tumors after different treatments. (*)p <
0.05, (**)p < 0.01. (h) Body weights were measured during the 17 day evaluation period in mice under the different conditions.
Dates indicate means and standard errors.

spectrum of HSA-ICG NPs was obtained with spectro-
meter (Figure 7a). As shown in Figure 7b, the tumor and
its margin, normal tissue sites were first labeled with
different color numbers. We detected the FL spectra
of ICG at normal tissue, tumor margin and tumor posi-
tion using spectrum-resolved technology (Figure 7c).
The ICG exhibited a characteristic emission spectra
with the peak located at 816 nm. The average FL
intensity of tumor positions is nearly 270 times than
that of normal tissue. The FL intensity collected from
the tumor margin (less than 1 mm from tumor) are still
19 times stronger than that of normal tissue, providing
excellent delineation of the tumor. Integrated FL in-
tensity of ICG at various locations was translated into a
mapping imaging using origin software (Figure 7d).
The results were indeed correlated with presence of
tumor and its margin, normal tissue as determined
with bright-field image. Therefore, in vivo NIR FL
imaging and HSA-ICG-based spectrum-resolved tech-
nology can precisely identifying tumor position, size
and margin, which are prone to guide focusing laser
beam on whole tumor tissue without damaging nor-
mal tissue.
Imaging-Guided In Vivo Synergistic Phototherapy. On the
basis of the treatment results in vitro, we expected to
apply HSA-ICG NPs for in vivo cancer phototherapy.
Under the guidance of NIR FL and PA dual-modal
imaging and spectrum-resolved technology, the laser
beam could be manipulated very precisely and flexibly,
and accurately focused on the whole tumor tissue
(Figure 8b). The real-time temperature change of mice
was analyzed using an infrared thermal camera, which
provided an effective tool for monitoring the treat-
ment process. As shown in Figure 8d, after the 5 min of
NIR laser irradiation, the tumors treated with PBS
and free ICG exhibited moderate increase to 37.8 and
40.6 °C, respectively. While the temperature of tumor
treated with HSA-ICG NPs increased rapidly to 57.2 °C,
which was high enough to ablate the malignant cells.
In order to confirm the PDT effect in vivo, the laser
irradiation was adjusted using the 5 min interval after
each minute of irradiation. The tumor exhibited a mild
temperature rise to 32.4 °C, which was insufficient to
irreversibly heat damage the tumor tissue. To deter-
mine the antitumor effect of free ICG and HSA-ICG NPs
mediated by heat or ROS, H&E staining of tumor

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Figure 9. In vivo cancer phototherapy in orthotopic mice models with 4T1 breast cancer. (a) IR thermal images of 4T1 tumor-
bearing mice exposed to 808 nm laser for 5 min (0.8 W/cm2). (b) Body weights were measured during the 18 day evaluation
period in mice under the different conditions. Dates indicate means and standard errors. (c) Tumour growth curves of
different groups of 4T1 orthotopic mice. (d) Survival rates of orthotopic mice bearing 4T1 tumors after different treatments.
(*)p < 0.05, (**)p < 0.01.

sections was performed. As shown in Figure 8e, in PBS
and free ICG treated groups, there was no obvious
tumor necrosis in histological section. On the contrary,
abundant karyolysis and sporadic necrotic was ob-
served in HSA-ICG NPs treated group with interval laser
irradiation (PDT), and apparent extensive cancer ne-
crosis occurred with continuous NIR laser irradiation
(PDT/PTT). The results indicated that simultaneous
PDT/PTT effect in vivo could be induced by single NIR
laser irradiation. HSA-ICG NPs can coordinate PDT with
PTT to get higher anticancer efficiency than single PDT
therapy in living mice.45
In order to further investigate the antitumor effi-
ciency of HSA-ICG NPs in mice bearing 4T1, the growth
rate of tumors was monitored every 3 days after treat-
ments (Figure 8f). The represent mice photos reflecting
the tumor size change showed in Figure S14. The
tumors treated with PBS and HSA-ICG NPs without
laser irradiation and free ICG plus laser irradiation grew
rapidly, indicating the 4T1 tumor growth was not
affected by HSA-ICG NPs, free ICG or laser irradiation.
The growth of 4T1 tumors was slightly inhibited by
HSA-ICG NPs plus interval and laser irradiation, which
indicated that the HSA-ICG NPs for PDT triggered by
interval laser irradiation could partly suppress tumor
growth, but tumor regrew after 3 days post-treatment.
The survival rate of this group was 40.0% on day 20
post-treatment (Figure 8g). When the continuous NIR
laser beam was accurately focused on the whole tumor
tissue (Figure 8b), the growth of 4T1 tumor was com-
pletely inhibited with 100% of survival rate on day 50,
and there was no tumor recurrence detected. The
simultaneous synergetic PDT/PTT therapy mediated
by HSA-ICG NPs plus single NIR laser could significantly
improve the phototherapeutic efficiency. During the
treatments, the body weight was monitored, which
indicated the treatments-induced toxicity (Figure 8h).
On day 17, the weight of the control groups treated
with PBS and HSA-ICG NPs in mice bearing 4T1 gradu-
ally increased by 6 and 11%, and those treated with
HSA-ICG NPs and free ICG plus continuous or interval
laser increased by 7, 8 and 11%. These groups were not
significantly different from the control group, suggest-
ing that the treatment was reasonably well-tolerated.
Moreover, the H&E staining images of major organs
collected from HSA-ICG NPs treated groups with NIR
laser showed that neither obvious damage nor inflam-
mation was observed compared to the control group
(Figure S15). The results indicated that the HSA-ICG NPs
possessed low cytotoxicity, and being suitable for
imaging-guided simultaneous synergistic PDT/PTT
therapy.
On the other hand, the continuous NIR laser beam
would be partly or over focused on the tumor tissue
without imaging-guided technology (Figure 8a and 8c).
In HSA-ICG NPs treated groups with partly laser
beam irradiation, the growth of 4T1 tumor was partly
inhibited, and regrow after 6 days post-treatment

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(Figure 8f). The survival rate of this group was 60% on
day 20 post-treatment (Figure 8g). On the contrary,
when the NIR laser beam was over focused on the
tumor position, the growth of 4T1 tumor was com-
pletely suppressed (Figure 8f). However, the body
weight of the treated mice decreased rapidly by 23%,
suggesting that the treatment has high side effects
(Figure 8g). The results demonstrated that the imaging-
guided phototherapy was prone to accurately focus
the NIR laser beam on the tumor and improve the
treatment efficiency and reduce treatments-induced
toxicity.
On the basis of the encouraging results, the HSA-
ICG NPs for synergistic phototherapy were further
investigated in an orthotopic breast cancer model.
The breast tumor grew on the mammary fat pad of
mice, keeping a distance from the surface skin.46 The
mice were treated when the tumor reached 40 mm3.
The results were shown in Figure 9. After the NIR laser
irradiation for 5 min, the tumors treated with HSA-ICG
NPs exhibited high increase to 53.8 °C, which was high
enough to ablate the malignant cells. The growth of
4T1 tumor in orthotopic mice models was completely
inhibited with 100% of survival rate on day 18, and
there was no tumor recurrence detected (Figure S16).
The results indicated that the HSA-ICG NPs could
MATERIALS AND METHODS
Materials. HSA was obtained from Beijing Biosynthesis Bio-
technology CO., Ltd. ICG, NaN3, GSH, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4,6-diamidino-
2-phenylindole (DAPI), and propidium iodide (PI) were
purchased from Sigma-Aldrich. Calcein-AM and Alexa Fluor
488 Annexin V/Propidium Iodide (PI) Cell Apoptosis Kit were
obtained from Invitrogen (USA). Phosphate-buffered saline
(PBS, pH 7.4), fetal bovine serum (FBS), RMPI 1640, trypsin-EDTA
and penicillin-streptomycin were purchased from Gibco Life
Technologies (AG, Switzerland). Ethanol was obtained from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ami-
con ultra-4 centrifugal filter with a molecular weight cutoff of
100 kDa was bought from Millipore (USA). All other chemicals
used in this study were of analytical reagent grade and used
without further purification. Superpure water (18.25 MΩ.cm,
25 °C) was used to prepare all solutions. BALB/c athymic nude
mice and BALB/c mice were maintained under aseptic condi-
tions in a small animal isolator. All food, water, bedding and
cages were autoclaved before use.
Determination of the Number of the Free Sulfhydryl. The number of
the free sulfhydryl of HSA, reduced HSA and HSA NPs was
determined using the Ellman's method.42 The preparation of
reduced HSA and HSA NPs was as follows. HSA was dissolved in
deionized water at concentration 40 mg/mL with 50 mM GSH at
room temperature to break up the intramolecular disulfide
bonds and expose free sulfhydryl groups. After 60 min, 2 mL
of solution were taken out to dialyze (membrane cutoff MW:
12 KD) in deionized water at 4 °C for 24 h to get rid of the
excessive GSH and probably its oxidized form GSSG. Then, 2 mL
of the desolvating agent ethanol was added into another 2 mL
of solution to supersaturate the HSA solution and precipitate
the NPs. The suspension was kept under stirring at room
temperature for 30 min. After that, the suspension was dialyzed
(membrane cutoff MW: 12
14 KD) with deionized water at 4 °C
for 24 h to remove ethanol and GSH.
SHENG ET AL.
ARTICLE
also be successfully applied for tumor phototherapy
in breast tumors in orthotopic mice models.
CONCLUSION
The present study reported a programmed assembly
strategy to fabricate HSA-ICG NPs successfully for
in vivo dual-modal imaging and tumor margin detec-
tion following PDT/PTT synergetic phototherapy. The
advantages of the prepared method are as follows: (1)
It could effectively maintain the biological activity of
HSA without using exogenous cross-linkers and toxic
chemicals; (2) The encapsulation of ICG in HSA NPs
remarkably improved the stability of ICG; and (3) HSA
NPs enhanced delivery of ICG into cancer cells. The
tumor, tumor margin and normal tissue could be
clearly detected using ICG-based in vivo FL and PA
dual-modal imaging and spectrum-resolved technol-
ogy, which was prone to avoid the effect of light
scattering and precisely guide cancer phototherapy.
The HSA-ICG NPs-mediated simultaneous synergetic
PDT/PTT treatments significantly improved the anti-
cancer effect, leading to superior tumor eradication
without any regrowth. Therefore, this novel theraonstic
nanoplatform can be a promising strategy for imaging-
guided synergetic cancer phototherapy, and is ex-
pected to have a great potential in clinical translation.
HSA-ICG NPs Generated by Programmed Assembly. 80 mg HSA and
20 mg ICG were first dissolved in 2 mL deionized water with
50 mM GSH at 37 °C for 1 h. Then, 2 mL of the ethanol was added
into the solution to precipitate the HSA-ICG NPs. The suspension
was kept under stirring at room temperature for 30 min. After
that, the suspension was dialyzed (membrane cutoff MW:
12
14 KD) with deionized water at 4 °C for 24 h to remove
ethanol, free ICG and GSH. To determine ICG loading in HSA-ICG
NPs, the HSA-ICG NPs solution was diluted in 5 mL of DMSO/H2O
(9:1, v/v) and sonicated for 30 min to extract ICG completely. ICG
levels were determined by UV/vis absorption spectra. ICG
loading was defined as ICG content (%, w/w) = (ICG weight in
HSA-ICG NPs/total HSA-ICG NPs weight)  100. All the measure-
ments were performed in triplicate.
Characterization of HSA-ICG NPs. The freshly obtained HSA-ICG
NPs suspension were transferred onto a 200 mesh copper grid
coated with carbon, stained with 2% (w/v) phosphotungstic
acid, dried at room temperature, then analyzed by TEM (FEI
Tecnai G2 F20 S-Twin, USA). UV/vis absorption spectra were
obtained with a PerkinElmer Lambda 25 UV/vis spectrophot-
ometer. The PL spectra were obtained by a FL spectrometer
(F900, Edinburgh Instruments Ltd.). Circular dichroism (CD)
spectra were recorded on an Applied Photophysis Chirascan
instrument at 25 °C. The size and zeta potential of the HSA-
ICG NPs were measured by dynamic light scattering (DLS), using
a Malvern Zeta Sizer (NanoZS). The samples have been
thoroughly purified with centrifugal filters from Millipore
(Centrifugal filter devices, 100 K) and dispersed in superpure
water prior to the measurements. Given the sensitivity of the
instrument, multiple runs (>3) were performed to avoid erro-
neous results.
Single Oxygen Detection. 20 ,70 -dichlorofluorescin diacetate
was employed to evaluate the singlet oxygen generation of
HSA-ICG NPs. A certain of HSA-ICG NPs was mixed with 20 ,70 -
dichlorofluorescin diacetate (1 μM) in water containing 2%
DMSO. The mixture solutions were irradiated with an NIR laser
VOL. 8 ’ NO. 12 ’ 12310–12322 ’ 2014 12319
www.acsnano.org

(808 nm, 1 W/cm2). The fluorescence was excited with a light
resource of 494 nm wavelength.
Cell Culture. The human embryonic kidney cell line 293T and
mouse breast cell line 4T1 were cultured in RMPI 1640 supple-
mented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin,
and 1% (v/v) streptomycin. Cells were incubated in a humidified
incubator at 37 °C with 5% CO2.
In Vitro Cell Uptake. For in vitro studies, 1  105 4T1 and 293T
cells were seeded on a glass-bottomed culture dish, respec-
tively. After 24 h, HSA-ICG NPs or free ICG was incubated with
different cells for 3 h at 37 °C. To remove the unbound con-
jugates, the cells were washed three times for 10 min by using
shaking incubation in Tris buffer and fixed with 3.7% formalde-
hyde solution (1 mL) and incubated at shaking incubation each
for 20 min. The nuclear dye DAPI was used as a positive control
to stain nuclei in the experiment. Images of cells were acquired
using a Leica DMI6000 inverted microscope with a Leica TCS SP5
confocal scanning system.
For cellular uptake experiment, the cells (1  105 cells per
well) were seeded in 6-well plates and incubated overnight, and
then incubated with 0.01 mg/mL HSA-ICG NPs or free ICG. After
incubation 3 h, cells were rinsed with PBS three times, tripsi-
nized, and resuspended with medium. Afterward, the cells were
collected by Accuir C6 flow cytometer using CFlow Plus soft-
ware (BD, Ann Arbor, MI).
DHE Staining. ROS formation was monitored by fluorescence
microscopy using dihydroethidium (DHE) as the indicator.
Briefly, 1  105 4T1 and 293T cells were seeded on a glass-
bottomed culture dish. After 24 h, HSA-ICG NPs was incubated
with different cells for 3 h at 37 °C. After the cells were washed
3 times using PBS, 0.2 mL of DHE solution (5 μM, PBS) was added
into the cells for 30 min at 37 °C. Afterward, the cells were rinsed
again with PBS, and subsequently illuminated using an 808 nm
laser with energy density of 1 W/cm2 for 5 min. Then, the nuclear
dye DAPI was used as a positive control to stain nuclei in the
experiment. Images of cells were acquired using a Leica
DMI6000 inverted microscope with a Leica TCS SP5 confocal
scanning system.
In Vitro Phototherapy. 4T1 cells (1 104 cells per well) were
seeded in 96-well plates and incubated overnight at 37 °C in
a humidified 5% CO2 atmosphere. After being rinsed with PBS
(pH 7.4), the cells were incubated with HSA-ICG NPs for 3 h at
37 °C under the same conditions. Afterward, the cells of experi-
mental group were rinsed again with PBS and immersed in
200 μL of fresh culture medium, and subsequently illuminated
using an 808 nm laser with energy density of 1 W/cm2 for 5 min.
The PDT effect of HSA-ICG NPs on 4T1 cells were further verified
by maintaining a constant temperature to avoid photothermal
effect, and the PTT effect of HSA-ICG NPs were further verified by
treating 4T1 cells with 100 mM NaN3 to avoid producing singlet
oxygen during laser irradiation. The laser spot was adjusted to
fully cover the area of each well. After illumination, cells were
incubated for 12 h in a 5% CO2, 95% air humidified incubator at
37 °C. Dark control group was kept under identical conditions as
the experimental group except for illumination. The standard
MTT assay was carried out to evaluate the cell viability.
The synergistic PDT/PTT effects of HSA-ICG NPs on 4T1 cells
were further verified using Calcein AM and propidium iodide
(PI) costaining. 4T1 cells (5  104 cells per well) were seeded in
6-well plates and incubated overnight at 37 °C in a humidified
5% CO2 atmosphere. After being rinsed with PBS (pH 7.4), the
cells were incubated with HSA-ICG NPs or free ICG for 3 h at
37 °C under the same conditions. Afterward, the cells of
experimental group were rinsed again with PBS and immersed
in 200 μL of fresh culture medium, and subsequently illumi-
nated using an 808 nm laser with energy density of 1 W/cm2 for
5 min. After another 4 h incubation cells were stained with
calcein-AM for visualization of live cells and with PI for visualiza-
tion of dead/late apoptotic cells, according to the manufac-
turer's suggested protocol (Invitrogen). Afterward, the cells of
experimental group were rinsed again with PBS and were
examined by eliminating the interference of background FL of
ICG with biological inverted microscope (Olympus IX71, JPN).
Animal Model. Animals received care in accordance with the
Guidance Suggestions for the Care and Use of Laboratory
SHENG ET AL.
ARTICLE
Animals. The procedures were approved by Shenzhen Institutes
of Advanced Technology, Chinese Academy of Sciences Animal
Care and Use Committee. Six-to-seven week old male BALB/c
athymic nude mice and BALB/c mice were maintained under
aseptic conditions in a small animal isolator and were housed in
a group of five in standard cages with free access to food and
water and a 12 h light/dark cycle. All animals acclimated to the
animal facility for at least 7 days before experimentation. BALB/c
mice subcutaneous tumor models were established by sub-
cutaneous injection of 1  106 4T1 cells onto the hind legs of
mice. BALB/c mice orthotopic tumor models were developed as
follow. 0.05 mL of the cell suspension was injected into the
mammary fat pad inferior to the nipple using a 28-gauge
needle. The breast tumor grew on the mammary fat pad of
mice, keeping a distance from the surface skin. All possible
parameters that may cause social stress, like group size, type
(treated and nontreated), etc., among the experimental animals
were carefully monitored and avoided. Animals were observed
daily for any behavioral abnormalities and weighed weekly.
Blood Circulation. To detect the blood circulation half-life of
HSA-ICG NPs, the BALB/c mice were randomly divided into 10
groups (n = 3 per group). Each group of mice was i.v. injected
with 100 μL of HSA-ICG NPs. The blood samples with ∼100 μL
were drawn from orbit of BALB/c mice postinjection of HSA-ICG
NPs at different time points (0.1, 0.2, 0.3, 0.5, 1, 3, 6, 8, 12, 24 h).
Each blood sample was dissolved in 900 μL of lysis buffer. The
concentration of HSA-ICG NPs in the blood was determined by
the FL spectrum of each solubilized blood sample using a FL
spectrometer. A series of dilutions of free ICG were measured to
obtain a standard calibration curve. Blank blood sample without
HSA-ICG NPs injection was measured to determine the blood
autofluorescence level, which was subtracted from the FL
intensities of injected samples during the concentration calcu-
lation. The HSA-ICG NPs are presented as the percentage of
injected dose per gram of tissue (% ID/g).
In Vivo Tumour Imaging and Tumour Margin Detection. Athymic
nude mice with subcutaneous 4T1 breast cancer xenografts
were used as the animal modal. The imaging studies were
performed when tumors reached ∼60 mm3. We intravenously
(i.v.) injected HSA-ICG NPs or free ICG (0.5 mg ICG/kg) into mice
(n = 3), and acquired FL images on CRI maestro system using a
704 nm excitation wavelength and a 735 nm filter at different
time points. The images were unmixed using the Maestro
software. Tumour-to-normal tissue ratio (T/N) was determined
and was expressed as mean ( SD. All mice were euthanized
after the 24 h imaging. Tumours as well as major organs were
collected and subjected to ex vivo imaging. The FL spectra of
HSA-ICG NPs from several locations, including the center of the
tumor, tumor margin and normal tissue, were examined by
using an NIR FL spectrometer (Inspector Series, DeltaNu). After
the spectra were acquired, the integrated signal intensity was
calculated. In vivo PA imaging was obtained with preclinical
photoacoustic computerized tomography scanner (Endra
Nexus 128, Ann Arbor, MI).
In Vivo Thermal Imaging. When the tumor size reached ∼60 mm3,
HSA-ICG NPs (2 mg ICG/kg) or free ICG (2 mg ICG/kg) was
intravenously injected into the tumor-bearing mice. Thermal
imaging was recorded by an infrared thermal imaging camera
(Ti27, Fluke, USA) when the tumors were exposed to 808 nm laser
of power density at 0.8 W/cm2.
In Vivo Synergistic Phototherapy. For synergistic phototherapy
studies, 35 mice bearing 4T1 tumors were randomly divided into
seven groups. The treatment scheme is as follows: (1) PBS, no irra-
diation; (2) HSA-ICG NPs, no irradiation; (3) free ICG (2 mg ICG/kg)
plus accurate irradiation (b); (4) HSA-ICG NPs (2 mg ICG/kg) plus
interval laser accurate irradiation (5 min interval after each
minute of irradiation) (b); (5) HSA-ICG NPs (2 mg ICG/kg) plus
part irradiation (a); (6) HSA-ICG NPs (2 mg ICG/kg) plus over
irradiation (c); (7) HSA-ICG NPs (2 mg ICG/kg) plus accurate
irradiation (b). The photoirradiation was applied 24 h after the
injection of HSA-ICG NPs or free ICG (808 nm, 0.8 W/cm2 for
5 min). The spot of laser beam was adjusted according to the
different experiment conditions. The tumor sizes and body
weights were inspected every 3 days. The tumor weight was
estimated using the formula, tumor volume = length  (width)2/2,
VOL. 8 ’ NO. 12 ’ 12310–12322 ’ 2014 12320
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assuming a tumor density of 1 mg/mL. The volume of tumors
was evaluated by normalizing the measured values. After
therapy, major organs as well as tumors were collected and
sectioned to 8 μm slices for H&E staining.
H&E Staining. H&E staining was performed according to a
protocol provided by the vendor (BBC Biochemical). Briefly,
8 μm cryogenic slides were prepared and fixed with 10% formalin
for about 30 min at room temperature. After washing with
running water for 5 min, the slides were treated with gradient
concentrations of alcohol (100, 95, and 70%), each for 20 s. The
hematoxylin staining was performed for about 3 min and
washed with water for 1 min. The eosin staining was performed
for about 1 min. The slides were washed, treated with xylene,
and mounted with Canada balsam. The images were acquired
on a Nikon Eclipse 90i microscope.
Conflict of Interest: The authors declare no competing
financial interest.
Acknowledgment. We are grateful to Dr. Junhui Zhai (United
Well Technology Limited (Shanghai)) and Professor Gang Liu
(Center for Molecular Imaging and Translational Medicine,
Xiamen University) for PA imaging studies. The authors grate-
fully acknowledge support for this research from the National
Natural Science Foundation of China (Grant No. 81301272,
81401521, 81071249, 81171446 and 81371679), Science and
Technology Foundation of Guangdong Province of China (Grant
No. 2009A030301010) and Shenzhen (JCYJ20130401170306862
and CXZZ20130506140505859), Key Project of Science and Tech-
nology of Guangdong (2012B090400043 and 2012B090600036),
Nanshan Innovative Technology Program (Grant No. KC2013-
JSJS0012A), Guangdong Innovation Team of Low-cost Health-
care, and SIAT Innovation Program for Excellent Young Research-
ers (201301).
Supporting Information Available: Supplementary methods
and results. This material is available free of charge via the
Internet at http://pubs.acs.org.
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